The aim of the proposed research is to determine the molecular mechanism of genetic regulation in a eucaryote. As a simple model system we are studying induction of Beta-galactosidase by lactose in the yeast Kluyveromyces lactis. Our experimental approach is to probe the mechanism of induction by genetic and biochemical techniques. A comparison of induction in mutant and wild type will be used to construct a genetic model of the regulatory process. In vitro studies with purified cell components will be used to verify the mechanism of regulation suggested by genetic and biochemical experiments. The cloned yeast Beta-galactosidase gene, isolated by recombinant DNA techniques, will be used as a probe: (1) to determine if transcription is regulated. Beta-galactosidase mRNA levels before and after enzyme induction will be measured using the cloned gene as a hybridization probe. (2) to determine the location and nucleotide sequence of transcription and translation initiation and termination sites and other regulatory regions. (3) to test for "splicing" of Beta-galactosidase mRNA. We will search for proteins that may be involved in the regulatory process by (1) comparing the protein composition of chromatin isolated from induced, uninduced, and mutant cells, (2) looking for proteins that bind specifically to nucleotide sequences preceding the coding region of the beta-galactosidase gene, and (3) investigating proteins that bind inducers. Factors that influence transcription of the beta-galactosidase gene will be examined using nuclei isolated from induced and uninduced cells. We will also reconstitute chromatin using the cloned beta-galactosidase gene and determine what factors affect its transcription.